Assembly of Hemoglobin Subunits: The kinetics of the formation of hemoglobin tetramer (alpha 2 beta 2) from isolated alpha and beta subunits will be determined with a stopped-flow apparatus. We will compare normal and variant subunits (such as beta S) and will also measure the formation of Hb F (alpha 2 gamma 2) and Hb A2 (alpha 2 delta 2) from their constituent subunits. The rate of assembly of hemoglobin tetramers in an erythroid precursor cell is likely an important determinant of the cell's hemoglobin phenotype. Molecular Basis of Sickling: The conformational requirements for polymerization of sickle hemoglobin will be studied by the substitution of manganese-heme in place of iron-heme. In these hybrid hemoglobins, the tertiary structure of the alpha or beta subunits can be selectively altered. The effect of these modifications on the solubility of deoxyhemoglobin S should provide valuable information on the conformational constraints underlying the polymerization of Hb S. The role of the red cell membrane in sickling will be evaluated by measuring the kinetics and solubility of deoxyhemoglobin S in the absence and presence of inside out and right side out vesicles. If the inner surface of the red cell membrane serves as a nucleation site for polymerization of Hb S, the delay time for gelation should be markedly shortened.